Authors:Boyu Li; Chengkun Liu; Fenglei Zhou; Xue Mao; Runjun SunPages: 279 - 284Abstract: Objectives To create a multifunctional medical material that combines the advantages of both nanofibers and macroyarns. Results A novel electrospinning-based approach was developed for creating polycaprolactone (PCL) nanofiber covered yarns (PCL-NCYs) in which polyglycolic acid multi-strand filaments (PGA-MFs) were used as the core. BALB/3T3 (mouse embryonic fibroblast cell line) cells were cultured on the PCL-NCYs substrate and cell morphology and proliferation were determined by methylthiazol tetrazolium (MTT) assay. Compared with PGA-MFs, PCL-NCYs had a higher porosity and tensile strength of 88 ± 8% and 348 ± 16 MPa and in particular, the porosity was four times higher. BALB/3T3 cells attached more easily onto the nanofiber structure and proliferated along the direction of nanofibers, indicating that PCL-NCYs can achieve better cell differentiation and proliferation. Conclusions PCL-NCYs can be created by combining electrospinning covering and textile twisting, and have better mechanical property and higher porosity, and can be used as a novel scaffold in tissue engineering.PubDate: 2018-02-01DOI: 10.1007/s10529-017-2466-3Issue No:Vol. 40, No. 2 (2018)

Authors:Arpana Kumari; Nitin Kishor; Purnananda GuptasarmaPages: 285 - 295Abstract: Objective To examine the potential for applications of TthLAC, a monomeric (~ 53 kDa) laccase encoded by the genome of Thermus thermophilus (strain HB 27) which can be produced at low cost in Escherichia coli. Result Functional, thermostable and mildly alkalophilic TthLAC of high purity (> 90%) was produced through simple heating of suspended (TthLAC overexpressing) E.coli cells at 65 °C. For reactions of short duration (< 1 h) the temperature for optimal activity is ~ 90 °C. However, TthLAC undergoes slow partial unfolding and thermal inactivation above 65 °C, making it unsuitable for long incubations above this temperature. With different substrates, optimal function was observed from pH 6 to 8. With the substrate, ABTS, catalytic efficiency (K m) and maximum velocity (Vmax) at 60 °C and pH 6.0 were determined to be 2.4 × 103 µM and 0.04 × 103 µM/min respectively. Ultra-pure, affinity-purified TthLAC was used to confirm and characterize the enzyme’s ability to oxidize known (laccase) substrates such as ABTS, syringaldazine and 4-fluoro-2-methylphenol. TthLAC decoloured up to six different industrial dyes, with or without the use of redox mediators such as ABTS. Conclusions Unlike versatile laccases from most other sources, which tend to be thermolabile as well as acidophilic, TthLAC is a versatile, thermostable, mildly alkalophilic laccase which can be produced at low cost in E.coli for various redox applications.PubDate: 2018-02-01DOI: 10.1007/s10529-017-2461-8Issue No:Vol. 40, No. 2 (2018)

Authors:Wei Wang; Yang Yu; Tong-Yi Dou; Jia-Yue Wang; Chenggong SunPages: 335 - 341Abstract: Objectives To screen the phylogenetically-nearest members of Cellulosimicrobium cellulans for the production of cellulosome-like multienzyme complexes and extracellular β-xylosidase activity against 7-xylosyltaxanes and to get corresponding molecular insights. Results Cellulosimicrobium (family Promicromonosporaceae) and all genera of the family Cellulomonadeceaec produced both cellulosome-like multienzyme complexes and extracellular β-xylosidase activity, while the other genera of the family Promicromonosporaceae did not. Multiple sequence alignments further indicated that hypothetic protein M768_06655 might be a possible key subunit. Conclusion This is the first report that many actinobacteria species can produce cellulosome-like multienzyme complexes. The production of cellulosome-like complexes and the extracellular β-xylosidase activity against 7-xylosyltaxanes might be used to differentiate the genus Cellulosimicrobium from other genera of the family Promicromonosporaceae.PubDate: 2018-02-01DOI: 10.1007/s10529-017-2469-0Issue No:Vol. 40, No. 2 (2018)

Authors:Klára Herkommerová; Jana Zemančíková; Hana Sychrová; Zuzana AntošováPages: 405 - 411Abstract: Objectives To improve the storage stability and reusability of various yeast strains and species by immobilization in polyvinyl alcohol (PVA) hydrogel particles. Results Debaryomyces hansenii, Pichia sorbitophila, Saccharomyces cerevisiae, Yarrowia lipolytica, and Zygosaccharomyces rouxii were immobilized in PVA particles using LentiKats technology and stored in sterile water at 4 °C. The immobilization improved the survival of all species; however, the highest storage stability was achieved for S. cerevisiae and Y. lipolytica which survived more than 1 year, in contrast to free cells that survived for only 3 months. Tests of the reusability of immobilized recombinant laccase-secreting S. cerevisiae revealed that the cells were suitable for repetitive use (55 cycles during 15 months) even after storage in water at 4 °C for 9 months. A suitable method for killing immobilized laccase-secreting cells without affecting the produced enzyme activity was also developed. Conclusions The immobilization of yeasts in PVA hydrogel enables long-term, cheap storage with very good cell viability and productivity, thus becoming a promising approach for industrial applications.PubDate: 2018-02-01DOI: 10.1007/s10529-017-2485-0Issue No:Vol. 40, No. 2 (2018)

Authors:Takuya Sugawara; Mariko Chinzei; Setsuko Numano; Chifumi Kitazaki; Munehiko AsayamaAbstract: Objective A novel filamentous cyanobacterium, a photosynthesizing microorganism, was isolated from a river, and its unique features of flocculation and pentadecane production were characterized. Results Microscopic observations and a phylogenetic analysis with 16S rDNA revealed that this strain was a Limnothrix species denoted as the SK1-2-1 strain. Auto cell-flocculation was observed when this strain was exposed to a two-step incubation involving a standing cultivation following a shaking preincubation. Flocculation was enhanced by blue light at a wavelength at 470 nm and irradiation for several hours to 1 day. Moreover, the strain exhibiting exponential cell growth may preferentially accumulate alkanes as pentadecane C15H32 alkane, which may be used as jet fuel, at a range of approximately 1% in the dry cell weight of flocculated cells. Conclusion This is the first study on biofuel production using flocculated cells in which the specific manner of production may be regulated by cultivation conditions.PubDate: 2018-03-05DOI: 10.1007/s10529-018-2525-4

Authors:Mengmeng Zhang; Mengjia Wang; Xiaocui Zhu; Wengong Yu; Qianhong GongAbstract: Objective To screen for the quorum-sensing (QS) inhibitors from marine-derived fungi and evaluate their anti-QS properties in Pseudomonas aeruginosa. Results QS inhibitory activity was found in secondary metabolites of a marine fungus Fusarium sp. Z10 using P. aeruginosa QSIS-lasI biosensor. The major active compound of this fungus was isolated by HPLC and identified as equisetin. Subinhibitory concentration of equisetin could inhibit the formation of biofilm, swarming motility, and the production of virulence factors in P. aeruginosa. The inhibition of las, PQS, and rhl system by equisetin were determined using Escherichia coli MG4/pKDT17, E.coli pEAL08-2, and E.coli pDSY, respectively. Real–time RT-PCR assays showed that equisetin could downregulate the mRNA expression of QS-related genes. Conclusions Equisetin proved its potential as an inhibitor against P. aeruginosa QS system and might also serve as precursor compound in development of novel therapeutics for infectious diseases by optimal design of structures.PubDate: 2018-03-03DOI: 10.1007/s10529-018-2527-2

Authors:Cahit Muderrisoglu; Sayit Sargin; Ozlem Yesil-CeliktasAbstract: Objective To improve the efficiency of reactions of β-glucuronidase (GUS)-assisted glucuronic acid (GluA) removal within a microfluidic system. Results β-glucuronidase from Helix pomatia was immobilised and characterised in silica-based sol–gel monoliths. Efficiency of the GUS-doped silica monoliths was tested for hydrolysis of p-Nitrophenyl-β-d-glucuronide (pNP–GluA) in both ml-scaled medium via batch reactions and microfluidic environment via continuous-flow reactions. In the microfluidic platform, within a duration of 150 min of continuous operation (flow rate: 1 µL/min), the obtained highest pNP yield was almost 50% higher than that of the corresponding batchwise reaction. However, increased flow rates (3, 5, and 10 µL/min) resulted in lower conversion yields compared to 1 µL/min. The microfluidic platform demonstrated continuous hydrolytic activity for 7 days with considerable reaction yields while using a small amount of the enzyme. Conclusion These results revealed that usage of the microreactors has considerable potential to efficiently obtain bioactive GluA-free aglycons from various plant-derived β-glucuronides for pharmaceutical applications. Graphical PubDate: 2018-03-01DOI: 10.1007/s10529-018-2530-7

Authors:Feng Liu; Yueping CaoAbstract: Objective To discover and isolate a glyphosate-resistant gene from a microorganism through gene mining. Results The full aroM gene from Acremonium sp. (named aroMA.sp.) was cloned using rapid amplification of cDNA ends. The transcriptional expression level of each domain increased significantly after glyphosate treatment in the aroMA.sp. complex and reached its maximum at 48 h. The aroA domain of the aroMA.sp. (named aroA A.sp.) was expressed in Escherichia coli BL21 (DE3) and the product was purified through Ni-NTA affinity chromatography. Furthermore, 45 KDa was indicated by SDS-PAGE and its enzyme activity was optimal at 30 °C and PH 7.0. The Ki/Km value of aroAA.sp. was 0.106, and the E. coli BL21 harboring aroAA.sp. could grow in the M9 minimal medium with 100 mM glyphosate. Conclusion The aroAA.sp. from the aroMA.sp. complex had high enzyme activity and glyphosate resistance. Therefore, this research offers a new strategy for improving glyphosate resistance using the aroA domain of the aroM complex in the fungi.PubDate: 2018-02-24DOI: 10.1007/s10529-018-2529-0

Authors:Young-Jun Choi; Hyemin Kim; Ji-Woo Kim; Seokjoo Yoon; Han-Jin ParkAbstract: Objectives The aim of the study is to generate a spherical three-dimensional (3D) aggregate of hepatocyte-like cells (HLCs) differentiated from human embryonic stem cells and to investigate the effect of the 3D environment on hepatic maturation and drug metabolism. Results Quantitative real-time PCR analysis indicated that gene expression of mature hepatocyte markers, drug-metabolizing enzymes, and hepatic transporters was significantly higher in HLCs cultured in the 3D system than in those cultured in a two-dimensional system (p < 0.001). Moreover, hepatocyte-specific functions, including albumin secretion and bile canaliculi formation, were increased in HLCs cultured in the 3D system. In particular, 3D spheroidal culture increased expression of CES1 and BCHE, which encode hepatic esterases (p < 0.001). The enhanced activities of these hepatic esterases were confirmed by the cholinesterase activity assay and the increased susceptibility of HLCs to oseltamivir, which is metabolized by CES1. Conclusions 3D spheroidal culture enhances the maturation and drug metabolism of stem cell-derived HLCs, and this may help to optimize hepatic differentiation protocols for hepatotoxicity testing.PubDate: 2018-02-20DOI: 10.1007/s10529-018-2528-1

Authors:Chih-Yao Chui; Pierre-Alexis Mouthuy; Hua YeAbstract: Objective To demonstrate that uniform poly(vinyl butyral) (PVB) fibres can be safely electrospun onto a monolayer of human dermal fibroblasts using a portable device. Results PVB in solvent mixtures containing various amounts of ethanol and water was electrospun. Six percent (weight-to-volume ratio) PVB in a 9:1 ethanol:water ratio was the solution with the highest content in water that could be electrospun into consistent fibres with an average diameter of 0.9 μm (± 0.1 μm). Four and five percent PVB solutions created beaded fibres. A 8:2 ethanol:water solution lead to microbead formation while a 7:3 ethanol:water mix failed to fully dissolve. The selected solution was successfully electrospun onto a monolayer of human dermal fibroblasts and the process had no significant effect (p < 0.05) on cell viability compared to the control without fibres. Conclusions PVB–ethanol–water solutions could be electrospun without damaging the exposed cell layer. However, further work is required to demonstrate the long-term effect of PVB as a wound healing material.PubDate: 2018-02-15DOI: 10.1007/s10529-018-2522-7

Authors:Fei Ge; Longbao Zhu; Anna Aang; Ping Song; Wanzhen Li; Yugui Tao; Guocheng DuAbstract: Yeast has been increasingly used as a host for the expression of enzymes. Compared to other expression systems, the yeast expression system has many advantages including its suitability for large-scale fermentation and its ability to modify enzymes. When expressed in yeast, many recombinant enzymes are N-glycosylated, and this may play an important role in their activity, thermostability and secretion. Although the mechanism underlying this process is not clear, the regulation of N-glycosylation by introducing or eliminating N-glycosylation at specific sites has developed into an important strategy for improving the production or catalytic properties of recombinant enzymes. In this review, we summarize the recent advances in understanding the effects of N-glycosylation on the expression and characteristics of recombinant enzymes, and discuss novel strategies for regulating N-glycosylation in yeast. We hope that this review will help improve the understanding of the expression and the catalytic properties of N-glycosylated proteins.PubDate: 2018-02-15DOI: 10.1007/s10529-018-2526-3

Authors:Kun He; Hong Sun; Hao SongAbstract: Objectives To enhance the yield of 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) from phytosterols, a phytosterol transport system was constructed in Mycobacterium sp. strain MS136. Results 9-OHAD can be produced via the controlled degradation of phytosterols by mycobacteria. This involves an active transport process that requires trans-membrane proteins and ATP. A phytosterol transport system from Mycobacterium tuberculosis H37Rv was constructed in Mycobacterium sp. strain MS136 by co-expression of an energy-related gene, mceG, and two integrated membrane protein genes, yrbE4A and yrbE4B. The resultant of the Mycobacterium sp. strain MS136-GAB gave 5.7 g 9-OHAD l−1, which was a 20% increase over 4.7 g l−1 by the wild-type strain. The yield of 9-OHAD was increased to 6.0 g l−1 by optimization of fermentation conditions, when 13 g phytosterols l−1 were fermented for 84 h in 30 ml biotransformation medium in shake flasks. Conclusions Phytosterol transport system plays an active role in the uptake and transport of sterols, cloning of the system improved the mass transfer of phytosterols and increased the production of 9-OHAD.PubDate: 2018-02-01DOI: 10.1007/s10529-018-2520-9